The processes of inflammation and oxidative stress are important in the pathogenesis of many diseases including cancer, Alzheimer's and Parkinson's diseases, asthma, atherosclerosis, diabetes, inflammatory bowel disease, multiple sclerosis, osteoarthritis and rheumatoid arthritis. Inflammation produces large amounts of reactive oxygen and nitrogen species that can induce oxidative damage to DNA and other cellular components; our bodies have evolved defensive mechanisms to protect our cells in addition to repair the DNA damage. Among the protective mechanisms are the various antioxidant scavengers and cytoprotective enzymes.
The expression of genes that encode these oxidative stress response enzymes are regulated through a cis-acting antioxidant response element (ARE) found in the promoter region of these genes. Nuclear factor E2-related factor 2 (Nrf2), a member of the Cap n'Collar (CNC) family of proteins, plays a major role in the oxidative stress response as the transcription factor that binds to the ARE to induce the coordinated expression of antioxidant and cytoprotective genes. It contains a highly conserved basic leucine zipper (bZIP) structure characteristic of the CNC transcription factors and requires a small Maf protein as a partner for nuclear transport and DNA binding. The interaction of Nrf2-Maf heterodimer with the cis-acting ARE is essential for the upregulation of oxidative stress response by Nrf2. Nrf2 is the central transcription factor that interacts with ARE to induce the expression of cytoprotective genes.
By comparing the amino acid sequences of Nrf2 across different species, 6 conserved homologous domains (Neh1 to Neh6 domains) of Nrf2 have been identified. Of these domains, the C-terminal Neh1 contains the bZIP domain for DNA binding with Maf, a nuclear transcription factor, and the N-terminal Neh2 domain acts as the regulatory domain by interacting with the cytoplasmic redox sensor protein, Keap1 (Kelch-like ECH-associated protein 1). Keap1 is a BTB-kelch protein consisting of five distinct domains: NTR (N-terminal region), BTB (broad complex, tramtrack, and brick a'brac), IVR (intervening region), DGR (double glycine repeat) or Kelch, and CTR (C-terminal region). The Kelch domain is a 6 bladed β-propeller in which each blade is comprised of four β-strands. Along with the CTR domain, the Kelch domain contributes to the complex formation with the N-terminal Neh2 domain of Nrf2. The BTB domain of Keap1 is a protein-protein interaction motif involved in the homodimer formation.
The N-terminal BTB domain of Keap1 interacts with Cul3, a member of the cullin family of E3 ubiquitin-protein ligases. Thus, Keap1 acts as the substrate adaptor protein that bridges the interaction between Cul3 and Nrf2 through its N-terminal BTB and the C-terminal Kelch domains. Normally, this Cul3-based E3 ubiquitin ligase complex functions to sequester Nrf2 in the cytoplasm by targeting multiple lysine residues in the Neh2 domain of Nrf2 for ubiquitination. The IVR domain of Keap1 contains a cysteine-rich region that acts as the primary sensor of oxidative response. Specifically, the modification of two highly reactive cysteine residues, C273 and C288, is believed to lead to the dissociation of Keap1-Nrf2 complex, the subsequent translocation of Nrf2 into the nucleus, and the ultimate activation of ARE genes. Disrupting the Keap1-Nrf2 complex may increase the body's defense against carcinogens and reactive oxygen species generated during oxidative stress by inducing the expression of ARE-regulated genes and upregulating the expression of oxidative stress response enzymes. Thus, the Keap1-Nrf2 complex in the ARE signaling pathway is believed to be the key target for cancer chemoprevention.
The DLG and the ETGE (SEQ ID NO:8) motifs within the Neh2 domain of Nrf2 are two evolutionary conserved motifs of CNC family of proteins. The DLG motif has been reported to be involved in ubiquitin-dependent degradation of Nrf2, while the ETGE motif has been reported to be essential for the Keap1 regulation of Nrf2 activity. Although both motifs bind to the same site in Keap1, the ETGE motif was reported to be approximately 100-fold higher in affinity to Keap1 than the DLG motif. Several different ETGE-containing peptides have been reported to displace Nrf2 from Keap1. The longer ETGE-containing 14mer and 16mer Nrf2 peptides (H-LQLDEETGEFLPIQ-OH (SEQ ID NO:10) and H-AFFAQLQLDEETGEFL-OH) (SEQ ID NO:7) were able to effectively displace the Nrf2 protein from the Keap1:Nrf2 complex; the binding affinity(K) between the longer Nrf2 peptides and the Keap Kelch domain was found to be as low as 20 nM as measured by isothermal titration calorimetry (ITC). The shorter 10mer Nrf2 peptide (H-LDEETGEFLP-OH) (SEQ ID NO:11) also displaces the Nrf2 protein from the complex, although it is much less effective than the longer Nrf2 peptides. With the exception of these Nrf2-derived peptides, there are currently no known small molecules that can directly interfere with the protein-protein interaction between Keap1 and Nrf2.